The present invention relates to a novel method of inserting viral DNA, which optionally may contain cargo-DNA, into plants or viable parts thereof, but preferably into plants of the monocotyledon class, and most preferably into plants of the family Gramineae, using suitable transfer microorganisms. Further comprised by the invention are recombinant DNA, plasmid and vector molecules suitably adapted to the specific conditions of the process according to the invention and the transgenic plant products obtainable in accordance with the said process.
In view of the rapid increase in world population and the associated greater need for food-stuffs and raw materials, increasing the yield of useful plants and also increased extraction of plant contents, that is to say progress in the field of foodstuffs and medicines, is one of the most urgent tasks of biological and biotechnological research. In this connection, for example the following should be mentioned as essential aspects: increasing the resistance of useful plants to diseases and pests or to unfavourable soil conditions, increasing resistance to plant-protecting agents such as insecticides, herbicides, fungicides and bactericides, and beneficially modifying the nutrient content or the yield of plants. Such desirable effects could in general be brought about by induction or increased formation of protective substances, valuable proteins or toxins and by interventions in the regulatory system of plant metabolism Influencing the plant genotype appropriately can be effected, for example, by transferring new genes into whole plants or into plant cells.
It has ready proved possible in many cases to insert selected DNA fragments into viral DNA and then, together with the virus, to introduce them into another organism. Although most plant viruses are transmitted under natural conditions by insects that feed on infected and uninfected plants, thereby causing fresh infection of plants, this route is too inconvenient and difficult to control to achieve a selective and systematic transmission of viruses. Thus, for example, specially bred insect populations would be required for such a method under contained conditions. In addition, it would be very difficult to achieve a controlled virus infection, especially of large amounts of plant material.
The mechanical inoculation of leaves with viruses, the method so far employed in genetic engineering, is of only limited applicability, as cloned viral DNA is commonly believed to be non-infectious.
Although it is possible to clone and study in bacteria a variety of types of viral genomes, for example single stranded DNA viruses which are obtained by cloning double-stranded DNA forms [Mullineaux, P. M. et al, 1984], many viruses that are cloned in bacteria cannot be reintroduced into plants or used for infecting plants. The use of methods such as in vitro mutagenesis and recombinant DNA technology are therefore ruled out in basic studies as well as for exploiting such viruses as carriers of selected foreign DNA. Such problems do not arise when using the method of this invention as set forth hereinbelow.
Prior to the present invention there have been only a few reports concerning the introduction of cloned viral DNA into plant cells.
Howell et al (1980), for example, describe infection of turnip plants by cloned CaMV DNA. It is specifically emphasized in the said reference that the cloned viral DNA must be excised from the recombinant plasmid before it is capable of infecting the turnip plants.
Lebeurier et al (1982) demonstrate that a cloned tandem dimer of CaMV DNA with a partial deletion is infectious in a plant assay. The viral genome was inoculated as part of a pBR322 double-stranded DNA plasmid by artificial leaf inoculation. Lebeurier et al do not teach introduction of a tandemly duplicated CaMV genome into the plant cell as part of an Agrobacterium Ti-plasmid using the Agrobacterium transformation system.
Cress et al (1983) demonstrate that dimeric PSTV cDNA is infectious in a plant assay when inoculated by artificial means as part of a recombinant bacterial plasmid. Again, Cress et al do not teach use of the Agrobacterium system as an alternative route for delivering the viral DNA into the plant cell.
Prior to the present invention the only mentioning of viral DNA in connection with the Agrobacterium transformation system can be found in U.S. Pat. No. 4,536,475 [Anderson] and Shewmaker et al (1985).
Anderson [U.S. Pat. No. 4,536,4751] discloses a variety of recombinant plasmid molecules which comprise a bacterial plasmid into which are ligated the border sequences from the T-DNA regions of the Ti-plasmid of Agrobacterium tumefaciens. Anderson teach that the CaMV DNA can be employed as a DNA source of an eucaryotic origin of replication, which was considered helpful in increasing the opportunity for integration of the introduced DNA to occur. Accordingly, Anderson disclose CaMV DNA sequences that are situated outside of the the T-DNA and thus have not been assigned for the introduction into the plant cell""s genome.
Shewmaker et al (1985), on the other side, describe experiments in which a full-length copy of CaMV is introduced into plant cells using a Ti-plasmid of Agrobacterium tumefaciens. However, within the genetic construct used by Shewmaker et al (1985) the full-length viral genome is broken in two places and could thus not give rise to viral infection. Accordingly, by the above experiments Shewmaker et al (1985) were only able to demonstrate that the introduced CaMV genome gave rise to two polyadenylation transcripts. The teaching of Shewmaker et al is thus confined to a showing that the two major promoters of the CaMV genome are supposedly able to function in plant cells.
The above short discussion of the cited references shows that the prior art teaches essentially two different experimental approaches.
The objective of the main approach, which is represented by the Lebeurier et al (1982), the Cress et al (1983), and the Howell et al (1980) reference, is to develop a plant viral transformation system which shall make use of the specific properties of infectious plant virus particles. To achieve this objective, either the cloned viral DNA is excised from the recombinant bacterial plasmid prior to infecting the plant material [Howell et al (1980); Cress et al (1983)], or the whole recombinant plasmid containing duplicated viral copies, which proved able to become recombined out in the plant cell, is introduced into the plant by artificial means [Lebeurier et al (1983)].
However, using the above roughly sketched experimental approach for developing a virus-based plant transformation system would not help to overcome the disadvantages which are involved in a pure viral vector system.
The second experimental approach, which is represented by Shewmaker et al (1983) and by U.S. Pat. No. 4,536,475 [Anderson], relates to studies for establishing novel, improved Ti-plasmid based vector systems, for example by use of plant viral regulatory DNA sequences, such as the two CaMV promoters described in Shewmaker et al (1983), or of the CaMV replication origin as described in U.S. Pat. No. 4,536,475 [Anderson].
Neither Shewmaker et al (1983) nor U.S. Pat. No. 4,536,475 [Anderson] teach the introduction of a complete, intact viral genome, which is capable of giving rise to a functional virus particle in the transformed plant.
Thus, it was one of the main objectives of the instant invention to provide a method for reintroducing cloned viral DNA, that is normally not infectious upon mechanical inoculation of plant material, into plants.
Within the scope of the present invention it was surprisingly found that in order to achieve this object the two principle experimental approaches discussed hereinbefore can be suitably combined. By taking a combination of selected and rather simple measures, parts of which were already known, it is possible to accomplish the transfer of a functional viral DNA to a plant.
This finding was very surprising, since it was long known that Agrobacterium-mediated DNA delivery is a very complex process involving complex DNA protein interactions, both on the bacterial and on the plant level which govern transmission of T-DNA to plant cells. So it is, for example, still not known what the T-DNA intermediates look like.
Agrobacterium carries three genetic components that are required for plant cell transformation. The T-DNA is the mobile DNA element that, unlike other transposable elements, does not encode the products that mediate its transfer. Instead, the Ti plasmid virulence region provides most of the trans-acting products for the DNA transfer to the plant. The third component of the T-DNA transfer process resides in the Agrobacterium chromosome.
The activation of the vir gene expression is followed by several dramatic changes to the T-DNA element on the Ti plasmid that ultimately result in its transfer to the plant cell.
Unlike DNA transfer between bacteria during conjugation, T-DNA must be capable of penetrating the plant cell membrane, localizing and eventually penetrating the nuclear membrane. For this purpose the T-DNA intermediate is expected to have a specific structure, the nature of which is not yet known. A considerable and meanwhile well recognized suggestion in this respect is that single stranded T-DNA intermediates are involved in the T-DNA transfer process [Zambryski A, (1988)], which would not leave any of the known options for the viral DNA to become released.
Therefore, owing to the many imponderabilities involved in DNA delivery via Agrobacterium it was very surprising to find that the concept envisaged by the present invention did actually work.
In particular, the present invention relates to a novel method of introducing cloned viral DNA or functional equivalents thereof, that are normally not infectious upon mechanical inoculation of plants, into plant material of plants that are naturally amenable, or, if not, are made amenable artificially, to transformation by a transfer microorganism such as, for example, a transfer microorganism of the genus Agrobacterium, or viable parts thereof such as, for example, plant cell culture cells, which method comprises essentially the following procedural steps:
a) inserting cloned viral DNA or functional parts thereof, that preferably are capable of giving rise to a systemic infection, and optionally may contain cargo DNA, into a T-replicon, for example a Ti-plasmid or Ri-plasmid of an Agrobacterium, in the vicinity of one or more T-DNA border sequences, the distance between said viral DNA and the T-DNA sequence or sequences being chosen such that viral DNA, including any cargo DNA present, is transferred to plant material;
b) introducing the replicon into a transfer microorganism, for example a microorganism of the genus Agrobacterium; and
c) infecting plant material with the transfer microorganism that has been modified in accordance with b).
This method ensures that, after induction of the microbial functions that promote the transfer of the plasmid DNA to plants, the inserted DNA is also transferred including any cargo DNA that may be present The transformed plant material so obtained can be regenerated to completely transformed plants. The said induction process described in detail below may, on the one hand, be triggered by the plant itself, if suitable culturing and application conditions are applied, the plant being stimulated thereby to synthesize the said inducers itself; or alternatively synthetic or natural inducers such as those provided in formulae I and Ia may be added to the culture medium individually or in combination in a suitable concentration.
In applying the method according to the invention it was further surprisingly found that not only those plants known to be host plants of Agrobacterium could be transformed by the method according to the invention, but also plants belonging to the monocotyledon class and here especially plants of the family Gramineae, which prior to the present invention were commonly believed to be insusceptible to an Agrobacterium infection.
The greatest problem in using recombinant DNA technology in plants from the monocotyledon group resides in the lack of suitable transfer microorganisms, with the aid of which transformation frequencies that are sufficiently high for practical application can be achieved and which could thus be used as auxiliaries for a specifically directed insertion into the plant genome. Agrobacterium tumefaciens, for example, one of the most used transfer microorganisms for inserting genetic material into plants, is excellently suitable for genetic manipulation of numerous dicotyledonous plants, but so far it has not been possible to achieve correspondingly satisfactory results with representatives of monocotyledons, especially monocotyledonous cultivated plants since, from the monocotyledon class, so far only a few selected families are known that respond to infection with Agrobacterium tumefaciens and thus, at least theoretically, might be open to genetic manipulation. These families are, however, from the point of view of agricultural economics, insignificant marginal groups which could at most, be of importance as model plants. [DeCleene M, 1985; Hernalsteens J P et al, 1984; Hooykaas-Van Slogteren G M S et al, 1984; Graves ACF and Goldman SL, 1987].
It is precisely the Gramineae family, however, which includes the cultivated plants that are the most important from the point of view of agricultural economics including our most important types of cereal, such as, for example, wheat, barley, rye, oats, maize, rice, millet, inter alia, which are of particular exonomic interest, so that the development of processes that make it possible, irrespective of the above-mentioned limitations, also to make Gramineae representatives open to direct genetic modification must be regarded as an urgent problem.
The present invention thus preferably relates to a method of introducing cloned viral DNA, that optionally may contain cargo-DNA and is normally not infectious upon mechanical inoculation of plant material into whole plants of the Gramineae family or into viable parts thereof.
Contrary to all expectations, in the course of the investigations carried out within the scope of this invention it has surprisingly been shown that in using the method according to the invention it is now also possible for plants from the monocotyledon group, but especially from the family Gramineae, to become transformed in a specifically directed manner using certain transfer microorganisms such as, for example, Agrobacterium tumefaciens, that is to say, now also important representatives from the monocotyledon group, especially cultivated plants belonging to the Gramineae family, are accessible to infection by the said transfer microorganism.
The plants transformed in this manner can be identified by suitable methods of verification. There has proved especially suitable for this the use of virus genomes of plant-pathogenic viruses, such as, for example, Maize Streak Virus (MSV), by means of which successful transformations can be verified very efficiently by way of the disease symptoms that appear.
However, within the scope of the present invention it was now surprisingly found, that by applying a combination of suitable procedural measures, involving, for example, the use of a suitable plant material and a suitable inoculation site for the DNA probe to be introduced, it is possible to achieve that high a transformation frequency, that it would no longer be necessary to rely on disease symptoms in order to verify a positive transformation event This would mean, however, that the Agrobacterium transformation system can now be applied directly to graminaceous monocots without any viral DNA being involved.
Attention is thus drawn especially to the broadening of the host spectrum of Agrobacterium tumefaciens to include Gramineae, by means of which even in representatives of this family a direct and specifically targeted manipulation of the genome is now possible.
In one of its aspects the present invention therefore relates to a process for inserting genetic material into monocotyledonous plants of the family Gramineae or viable parts thereof, wherein a transfer microorganism that contains the genetic material in a transportable form is made usable for infection of monocotyledons by employing suitable culturing and application methods that make possible the induction of the virulence gene functions of the transfer microorganism, and wherein monocotyledonous plants or viable parts thereof are infected therewith.
Further comprised by the invention are recombinant DNA, plasmid and vector molecules suitably adapted to the specific conditions of the process according to the invention and the transgenic plant products obtainable in accordance with the said process.